The present invention relates generally to multi-well plates and to their use in conjunction with infrared spectrum imaging systems.
In assay screening, a large number of cellular events (e.g., calcium flux, etc.), physiological events and/or molecular events (e.g., chemical reactions, etc.) are monitored and analyzed. These events, hereinafter referred to as xe2x80x9ctarget events,xe2x80x9d are often carried out in multi-well (e.g., micro-titer) plates. As the name xe2x80x9cmulti-wellxe2x80x9d implies, these plates contain a multiplicity of wells (96-well, 384-well, 1536-well plates are typical) that are organized in a two-dimensional array. The wells are quite small, having a diameter that is typically in a range of about 1 millimeter to about 6 millimeters as a function of the number of wells in the plate.
Due to the large number of events taking place in the wells, time-consuming methods that directly examine each well (e.g., microscopic examination, etc.) are unsuitable for data acquisition. To screen such a large number of events, a xe2x80x9csnap shotxe2x80x9d of the whole plate is advantageously taken using various imaging techniques.
Perhaps the most common imaging techniques are those that image visible spectrum light, such as fluorescence imaging and luminescence imaging. In the former process, when an event of interest (e.g., a cellular event, physiological process, chemical reaction, etc.) occurs, a detection reagent emits light (i.e., fluoresces) when excited by an appropriate excitation source (e.g., ultraviolet light). The emitted light, which provides qualitative and/or quantitative information about the event, is captured and converted to electrical signals using, for example, a charge coupled device (xe2x80x9cCCDxe2x80x9d). The CCD comprises an array of thousands of cells that are capable of receiving light from multiple wells at the same time. The signals are analyzed, via suitable software, to recover information concerning the event. Luminescent imaging (chemi- or bio-) is similar to fluorescence imaging, except that excitation light is not required.
Area or array-type fluorescent imaging devices are very complex and, hence, very expensive (c.a., $100,000 to $400,000). These imaging devices typically include an excitation light source, complicated optics, filters, a CCD, a cooler for the CCD, a control unit, software, positioners, and other elements. While an excitation light source is not required for luminescence imaging, many of the luminescent reactions are so dim that a highly optimized imaging system, including the most sensitive form of cooled CCD camera and very efficient lenses, are required.
In addition to the high cost of such imagers, fluorescence and luminescence imaging is complicated by the requirement of a suitable detector reagent. While specific detector reagents have been developed for various applications, there are no universally applicable reagents.
Consequently, a less costly and less complicated alternative to visible spectrum (i.e., fluorescence and luminescence) imaging is desirable. One possible alternative is thermal or infrared imaging, wherein a change in energy that accompanies every chemical reaction and physiological process is monitored to obtain useful information. The energy change is observed as a temperature change within the wells of the multi-well plate.
Temperature changes that are being monitored are often quite small. In fact, temperature changes due to reaction and physiological processes can be significantly smaller than temperature changes due to incidental processes, such as evaporation. Furthermore, infrared radiation that is emitted from nearby objects can propagate into the wells of the multi-well plate and be imaged by the detector, swamping any temperature changes that might have occurred due to reaction, or at least introduce error into the measurements.
Consequently, there is a need for a way to improve the sensitivity or otherwise increase the signal-to-noise ratio of infrared imaging processes.
The problems described above related to infrared imaging are ameliorated by a multi-well plate that includes an infrared radiation (xe2x80x9cIRxe2x80x9d) reflective coating over the surface of the wells, in accordance with the illustrative embodiment of the present invention. In some embodiments, the reflective coating comprises a layer of a metal, such as gold, copper or the like.
The reflective coating provides at least two benefits. One benefit is that more of the IR radiation that is generated by target events is reflected toward the IR detector. A second benefit is that, with an appropriately deep well, stray IR that is introduced into a well from external sources is substantially totally internally reflected by the IR reflective coating. Consequently, such stray IR is not imaged, does not appear as noise and does not interfere with the measurement of temperature changes that are due to the target events being monitored.
In some multi-well plates described herein, the wells have a parabolic or near parabolic shape. Substantially all of the IR that is emitted at or near the focal point of such wells is reflected out of the mouth of the well (i.e., toward the IR detector) rather than dissipating into the plate. The parabolic shape of the well collimates IR emitted from the focal point such that path of radiation out of the well is normal to the surface of the multi-well plate and normal to the IR detector. Consequently, parabolic-shaped wells reduce the incidence of xe2x80x9cspilloverxe2x80x9d wherein IR is detected by a neighboring detector element (or group of detector elements) that are xe2x80x9cassignedxe2x80x9d to detect the IR emitted from neighboring wells.
Improved infrared imaging systems in accordance with the illustrative embodiment of the present invention incorporate the multi-well plates described herein. And, as a consequence, some of the IR imaging systems described herein exhibit a higher signal-to-noise ratio than some prior art IR imaging systems.
One infrared imaging system described herein comprises a multi-well plate as described above, a multi-well plate holder for supporting the multi-well plate, an isothermal chamber that receives the multi-well plate holder and the multi-well plate, an infrared camera that is focused on the multi-well plate and signal processing electronics that are electrically connected to the infrared camera and that are operable to receive a signal that is generated thereby.
A second infrared imaging system described herein comprises a multi-well plate as described above, and an IR detector in the form of a focal plane array that is disposed in parallel, opposed and aligned relation therewith. In some variations, the IR detector and the multi-well plate are separated by a space that is about one millimeter or less.
A third infrared imaging system described herein comprises a multi-well plate as described above and a detector plate. The detector plate comprises a plurality of wells having a parabolic shape, an IR-reflective coating disposed on the wells, and a sensor element that is disposed at a focal point within the wells. In some variations, the multi-well plate is separated from the detector plate by a space that is about one millimeter or less.
A fourth infrared imaging system described herein comprises a multi-well plate and a reflector that abut one another in opposed and aligned relation. The wells in the multi-well plate have an IR reflective coating and a hemispherical or truncated hemispherical shape. In some embodiments, the reflector comprises a plurality of wells, each having a hemispherical shape and having an IR reflective coating. When the multi-well plate and the reflector are brought together, wells aligned, each pair of aligned wells forms a substantially spherical enclosure. Each spherical enclosure is provided with a xe2x80x9cradiation-transparent windowxe2x80x9d through which emitted IR escapes from the enclosure. The radiation-transparent window is the only location at which emitted IR can escape from the enclosure. The radiation-transparent window leads to an IR sensor element.
A method in accordance with the illustrative embodiment of the present invention comprises: emitting IR near a focal plane, receiving the emitted IR at a first surface and reflecting it therefrom in a direction that is substantially perpendicular to the focal plane, receiving the reflected IR at a second surface and reflecting it therefrom to a focal point and detecting said infrared radiation at said focal point.